猪传染性胃肠炎病毒双抗体夹心ELISA检测方法的建立

Development of a double antibody sandwich ELISA for detection of transmissible gastroenteritis virus

为建立猪传染性胃肠炎病毒(TGEV)的快速检测方法,本研究以抗TGEV N蛋白单克隆抗体(MAb)为包被抗体,纯化的抗TGEV N蛋白多克隆抗体(PcAb)为检测抗体建立了TGEV双抗体夹心ELISA方法,优化其反应条件为:抗TGEV N蛋白MAb包被浓度为0.4μg/mL,抗TGEV N蛋白PcAb和酶标二抗的工作浓度分别为3.5μg/mL和1∶5 000,以P/N>2,同时OD490nm≥0.316作为阳性判定标准。该ELISA的重复性变异系数小于10%,与猪流行性腹泻病毒、猪细小病毒、猪瘟病毒、猪轮状病毒等无交叉反应。对22份临床猪粪便样品RT-PCR的阳性检出率为31.8%;ELISA的阳性检出率为22.7%,二者符合率达81.8%,结果表明本研究建立的双抗体夹心ELISA方法检测TGEV具有较高的特异性和敏感性,可以用于TGEV的病原学检测。

To develop a method for the detection of porcine transmissible gastroenteritis virus （TGEV）, a double-antibody sandwich ELISA （DAS-ELISA） was developed using monoclonal antibody （MAb） and purified polyclonal antibody against viral N protein as capture and detector antibody, respectively. The optimized reaction conditions included coating with 100 p^L/well of MAb at concentration of 0.4 Izg/mL, probing with the working concentration of 3.5 tzg/mL purified IgG from the polyclonal antibody and judging with P/N〉2 and OD^o~〉 0.316 as positive criteria. The DAS-ELISA was specific detection of TGEV, but no cross-reaction with classical swine fever virus, porcine epidemic diarrhea virus, porcine rotavirus, and the intra-assay and inter-assay coefficient of variability were within 10%. In addition, a total of 22 clinical fecal samples were tested by the DA$-ELISA and PCR, and the coincidence was 81.8% with the positive rates of 31.8% and 22.7%, respectively. These data demonstrated that the DAS-ELISA was sensitive and specific which provided a useful tool for diagnosis of TGEV infection.